Apoptosis is a form of cell death mediated by an intracellular program. The intracellular program proceeds through a series of biochemical events that result in various changes to cell morphology, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and DNA fragmentation. Proteolytic enzymes termed caspases are thought to be primarily responsible for these hallmarks of apoptosis.
Apoptosis is fundamental to various biological processes, such as adult homeostasis (i.e., to keep the number of cells relatively constant), response to injury (i.e., to kill damaged cells), development (e.g., to help pattern developing tissues), and maturation of the immune system (e.g., to remove lymphocytes that are ineffective and/or harmful), among others. Accordingly, many diseases are associated with a change in the rate of apoptosis. For example, tumor cells may have a resistance to apoptosis that provides a selective growth advantage over normal cells. In contrast, unwanted apoptosis may cause the neurological damage prevalent in various neurological diseases, such as stroke, Parkinson's disease, and Alzheimer's disease. Members of the Bcl-2 family regulate activation of the caspases. This family consists of two groups, namely, pro-death proteins (e.g., Bax) and pro-survival proteins (e.g., Bcl-2). The ratio of pro-death to pro-survival proteins within a cell generally determines whether the cell undergoes apoptosis or survives.
Bim protein is a pro-death member of the Bcl-2 family. The Bim protein is expressed as at least three protein isoforms of different length (i.e., BimEL, BimL, and BimS) having distinct potencies for promoting cell death, with the BimS protein isoform being most potent. The Bim protein plays a critical role in mediating cell death in various cell types. Therefore, drugs that modify levels of Bim protein would facilitate treating diseases characterized by abnormally low or abnormally high levels of apoptosis.
The steady-state level of a protein within a cell is generally determined by the rate at which the protein is synthesized relative to the rate at which the protein is degraded (catabolized). The primary mechanism for protein catabolism in cells is the Ubiquitin Proteasome Pathway (UPP). Protein catabolism by the UPP involves two successive steps: (1) covalent attachment of multiple ubiquitin polypeptides to a protein substrate (“ubiquitination”) to produce a ubiquitin-tagged protein; and (2) degradation of the ubiquitin-tagged protein by the 26S proteasome.
FIG. 1 shows a schematic flowchart 50 representing selected aspects of the UPP, including a ubiquitination portion 52 and a degradation portion 54. In the ubiquitination portion, an E3 ubiquitin ligase 56 (also termed an “E3 ligase”) promotes conjugation of a ubiquitin polypeptide 58 to a protein substrate 60. In particular, the ubiquitin polypeptide is transferred to the protein substrate from an E2 ubiquitin-conjugating enzyme 62 (also termed an “E2 enzyme”), which serves as a ubiquitin donor. Before this transfer, in an earlier step not shown here, the E2 enzyme receives ubiquitin polypeptide 58 in an activated form from an E1 ubiquitin-activating enzyme. Then, E3 ligase 56 targets transfer, indicated by an arrow at 64, of ubiquitin polypeptide 58 from E2 enzyme 62 to protein substrate 60. To target this transfer of ubiquitin, the E3 ligase may interact with both the E2 enzyme and the protein substrate at the same time, as shown here, or may interact sequentially such that ubiquitin is received first by the E3 ligase from the E2 enzyme and then is transferred to the protein substrate. In any event, the transfer of a ubiquitin polypeptide to the protein substrate may be performed repeatedly, indicated at 66, to form a ubiquitin-tagged product 68 in which protein substrate 60 is conjugated to a plurality of ubiquitin polypeptides 58. In degradation portion 54 of FIG. 1, ubiquitin-tagged product 68 may be recognized and processed by the 26S proteasome to produce degradation products 70 and released ubiquitin polypeptides 58.
A cell may contain only a few types of E1 enzymes, a larger number of E2 enzymes, and an even greater diversity of E3 ligases. Consistent with this high diversity of E3 ligases employed by the cell, each E3 ligase is thought to play a primary role in substrate identification for degradation in the UPP. However, for most proteins, a corresponding E3 ligase that targets degradation has not yet been identified.
Bim protein has been reported to be ubiquitinated, which suggests that Bim protein is degraded in the Ubiquitin Proteasome Pathway using a Bim-selective E3 ligase. Furthermore, phosphorylation of Bim protein on Ser65 in humans (Ser69 in rat) by p42/p44 MAP kinase (MAPK) has been shown to be an essential step for Bim ubiquitination and proteasomal degradation. As a result, mitogen stimulation of cells, which increases the level of active MAP kinase, may produce increased Bim ubiquitination and degradation, and thus less Bim protein and increased cell survival. However, a Bim-selective E3 ligase that is MAPK-dependent for interaction with Bim protein has not been identified in the prior art, although such an E3 ligase would provide a valuable tool for drug design, clinical diagnostics, and treatment of disease.